Event: 396

Key Event Title


Covalent Binding, Protein

Short name


Covalent Binding, Protein

Biological Context


Level of Biological Organization

Cell term


Cell term
eukaryotic cell

Organ term


Key Event Components


Process Object Action
protein binding electrophilic reagant increased

Key Event Overview

AOPs Including This Key Event


AOP Name Role of event in AOP
Skin Sensitisation AOP MolecularInitiatingEvent
Respiratory Sensitisation/Allergy induced by covalent binding to proteins MolecularInitiatingEvent



Taxonomic Applicability


Term Scientific Term Evidence Link
human Homo sapiens NCBI
guinea pig Cavia porcellus NCBI
mouse Mus musculus NCBI

Life Stages


Sex Applicability


Key Event Description


The molecular initiating event is covalent binding of electrophilic chemical species with selected nucleophilic molecular sites of action in proteins generating immunogenic neoantigens through a process termed haptenisation[1];[2]. In contrast to receptor-mediated chemical interactions electrophiles are not specific with regard to their molecular target. Moreover, some chemicals are able to react with several different nucleophilic chemical substituents. Therefore, the identification of the specific target protein is not considered to be critical. Moreover, it is recognized that reactivity measured with a particular nucleophilic target or model nucleophile does not necessarily reflect a specific chemical reaction, as many reactions target the same chemical substituent[3]. For toxicological endpoints for which protein binding is important, the biological nucleophile is assumed to be selected amino acids. The exact extent of adduct formation to each amino acid is dependent on the relative hardness / softness of the electrophile and nucleophile[3]. The inability to identify the exact biological nucleophile is deemed less important than information regarding the electrophile. As noted in the hard-soft acid base theory, a soft electrophile will have a relative preference for a soft nucleophile; while a hard electrophile will have a relative preference for a hard nucleophile. As a consequence, for a series of electrophiles assigned to the same mechanistic cluster within a particular domain, the relative rates of reactivity between each electrophile and any nucleophile will remain the same. In other words, while absolute reactivity may vary with protocols, relative reactivity will usually not vary significantly[3]. Binding experiments with small model nucleophiles reveal that, within a particular reaction within a mechanism, the rate of reactivity varies markedly. Moreover, while some compounds appear to bind exclusively with thiol or amine, others bind to a variety of nucleophiles. However, an electrophile is most likely to exhibit a preference for a particular nucleophile. In more complex systems, nucleophilic target preferences may be masked by other factors. It is self-evident that the number of cysteine and lysine residues within a protein will impact target probability. For example, for serum albumin, a major serum protein, 10% of the amino acid residues are lysine but albumin has very few free cysteine residues. Also, it is self-evident that a target site (e.g. cysteine or lysine) which is located on an exposed surface of a protein is more likely to react with an electrophile than one that is located within a grove or fold of a protein. Such steric constraints are imposed by the primary structure (i.e. amino acid sequence) of the peptide or protein, as well as the secondary and tertiary structure of proteins imposed by disulfide bridges, and folding and coiling. Similarly, the microenvironment of the reaction site (e.g. hydrophilic versus hydrophobic) may affect the probability of a particular reaction. Free cysteine residues are more abundant in proteins in the aqueous cytosol than in the non- aqueous biomembranes [4]. An ancillary event in identifying protein-binding is metabolism and/or abiotic transformation (e.g. autoxidation)[5].

How It Is Measured or Detected


In silico models, including physiological-based pharmacokinetic models and traditional structure activity ones, as well as in vitro and in vivo experimental approaches exist.

In silico Methods

It is generally recognized that reaction-based methods, as opposed to other means of defining chemical similarity, allow for easier interpretation and provide greater confidence in their use[6]. Chemical reactions related to covalent protein binding have recently been reviewed[7];[8];[9]. Measurements and estimations of reactivity have also recently been reviewed[1];[3]. Computational or in silico techniques to predict chemical reactivity have been developed; they vary in complexity from the relatively simple approach of forming chemical categories from 2D structural alerts (i.e. SARs for qualitative identification of chemical sub-structures with the potential of being reactive), such as used in the Organisation for Economic Co-Operation and Development (OECD)QSAR Toolbox[10] to QSAR models (i.e. quantitative prediction of relative reactivity) as described by Schwöbel et al.[11].

In Chemico Protocols and Databases

While methionine, histidine, and serine all possess nucleophilic groups that are found in skin proteins, the –SH group of cysteine and the ε-NH2 group of lysine are the most often studied. Soft electrophilic interactions involving the thiol group can be modelled with small molecules. Glutathione (GSH; L-γ-glutamyl-L-cysteinyl-glycine) is the most widely used model nucleophile in soft electrophilic reactivity assays. Typically, chemicals are incubated with GHS and, after a defined reaction time, the concentration of free thiol groups is measured. Such depletion based assays assume adduct formation, which is typically not confirmed. Good relationships between GSH reactivity and toxicity have been demonstrated. Examples of this method can be found in the literature[3];[12];[13];[14]. Recently, OECD adopted the new Test Guideline (TG) No442C: In chemico skin sensitisation – Direct Peptide Reactivity Assay (DPRA). This method quantifies the reactivity of chemicals towards model synthetic peptides containing either lysine or cysteine[15]. The DPRA protocol can be found in the EURL ECVAM Database Service on Alternative Methods to animal experimentation (DB-ALM): Protocol No154 for Direct Peptide Reactivity Assay (DPRA) for skin sensitisation testing[16]. The importance of reaction chemistry for sensitisation indicates that identification of the reaction limited chemical spaces is critical for using the proposed AOP. Systematic databases for reaction-specific chemical spaces are being developed. For example, in chemico databases reporting measurements of reactive potency currently exist for Michael acceptors ([14];[17];[18]). The use of model nucleophiles containing primary amino (–NH2) groups, such as in the amino acids lysine are less well-documented, with the principle of measuring relative reactivity being the same as for thiol[1].

Respiratory Sensitizers

Both respiratory and skin sensitizers are detected by in vitro and in silico methods used to measure electrophilic binding to proteins and peptides. (Basketter et al., 2017) The rate of covalent binding can also be measured. (Natsch and Gfeller, 2008) Dik et al. modified the DPRA protocol to include two peptide depletion measurement time points, and added high-performance liquid chromatography mass spectrometry (MS) analysis of reaction products, which improved predictive capacity. (Dik et al., 2016) Other authors have worked to investigate the binding of diisocyanates in vapor and liquid phases with LC/MS, MS/MS, and ELISA, as well as, Western blot. (Wisnewski et al., 2013a, 2013b, Hettick et al., 2012, Hopkins et al., 2005, Hettick and Siegel, 2011)

Overview table: How it is measured or detected

Method(s) Reference URL Regulatory


Validated Non


Direct Peptide Reactivity Assay (DPRA) TG 442C [1] X X  
DB-ALM [2]

Domain of Applicability


The OECD 2012 document does not indicate in vivo assays that measure covalent protein binding.


Evidence for Perturbation by Stressor

Overview for Molecular Initiating Event


The in chemico, in vitro, and in vivo experimental evidence is logical and consistent with the mechanistic plausibility proposed by covalent reactions based on the protein binding theory ([1];[19];[20]). In selected cases, (e.g. 1-chloro-2,4-dinitrobenzene) where the same compound has been examined in a variety of assays (see Annex 1 of[21]), the coherence and consistency of the experimental data is excellent. Alternative mechanism that logically present themselves and the extent to which they may distract from the postulated AOP. It should be noted that alternative mechanisms of action, if supported, require a separate AOP. While covalent reactions with thiol groups and to lesser extent amino groups, are clearly supported by the proposed AOP, reactions targeting other nucleophiles may or may not be supported by the proposed AOP. Limited data on chemical reactivity shows that two competing reactions are possible, the faster reaction dominates. However, this has yet to be proven in vitro or in vivo.

Earlier work on the molecular basis of skin sensitisation was reviewed by Lepoittevin et al. (1998)[22], since then our knowledge of skin sensitisation has continued to expand. Recent reviews (see[3];[9];[20];[22];[23];[24];[25]) repeatedly stress the same key steps leading to sensitisation. These events include hapten formation (i.e., the ability of a chemical to react with skin proteins).

The binding behavior of diisocyanates in particular has been well studied. Wisnewski et al.29,30 demonstrate that hexamethylene diisocyanate (HDI) and 4,4’-diphenylmethane diisocyanate (MDI) react with glutathione (GSH) across an in vitro physiologically relevant vapor/liquid-phase barrier to form conjugates, which may ‘‘shuttle,’’ via a carbamoylating reaction, the chemical to bind with serum albumin. Diisocyanates (MDI) react with GSH across an in vitro physiologically relevant vapor/liquid-phase barrier to form conjugates, which may ‘‘shuttle,’’ via a carbamoylating reaction, the chemical to bind with serum albumin.

In contrast to skin sensitization where cysteine and lysine are both key nucleophiles, experimental work has suggested that some respiratory sensitizers appear to preferentially bind to lysine; (Hettick et al., 2012, Lalko et al., 2012, Holsapple et al., 2006, Hopkins et al., 2005) however, an in chemico analysis of a larger set of respiratory sensitizers indicates lack of a simple division between the reactivity preferences of the two types of sensitizers, showing that certain classes displayed a lysine preference, for example, anhydrides, whereas others, such as diisocyanates, do not. (Dik et al., 2016)

While respiratory sensitizers and skin sensitizers can both bind to cellular and serum proteins in separate cultures, a study comparing the binding profiles of both classes in co-culture systems found that skin sensitizers preferentially bind cellular proteins, while respiratory sensitizers preferentially bind serum proteins. (Hopkins et al., 2005)



  1. 1.0 1.1 1.2 1.3 Gerberick F, Aleksic M, Basketter D, Casati S, Karlberg AT, Kern P, Kimber I, Lepoittevin JP, Natsch A, Ovigne JM, Rovida C, Sakaguchi H and Schultz T. 2008. Chemical reactivity measurement and the predictive identification of skin sensitisers. Altern. Lab. Anim. 36: 215-242.
  2. Karlberg AT, Bergström MA, Börje A, Luthman K and Nilsson JL. 2008. Allergic contact dermatitis- formation, structural requirements, and reactivity of skin sensitizers. Chem. Res. Toxicol. 21: 53-69.
  3. 3.0 3.1 3.2 3.3 3.4 3.5 Schwöbel JAH, Koleva YK, Bajot F, Enoch SJ, Hewitt M, Madden JC, Roberts DW, Schultz TW and Cronin MTD. 2011. Measurement and estimation of electrophilic reactivity for predictive toxicology. Chem. Rev. 111: 2562-2596.
  4. Hopkins JE, Naisbitt DJ, Kitteringham NR, Dearman RJ, Kimber I and Park BK. 2005. Selective haptenation of cellular or extracellular proteins by chemical allergens: Association with cytokine polarization. Chem. Res. Toxicol. 18: 375-381.
  5. Lepoittevin JP. 2006. Metabolism versus chemical transformation or pro-versus prehaptens? Contact Dermatitis 54: 73-74.
  6. Freidig AP and Hermens JLM. 2001. Narcosis and chemical reactivity QSARs for acute toxicity. Quant. Struct. Act. Rel. 19: 547-553.
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  8. Enoch SJ, Ellison CM, Schultz TW, Cronin MTD. 2011. A review of the electrophilic reaction chemistry involved on covalent protein binding relevant to toxicity. Crit. Rev. Toxicol. 41: 783– 802.
  9. 9.0 9.1 OECD 2011. Report of the Expert Consultation on Scientific and Regulatory Evaluation of Organic Chemistry-based Structural Alerts for the Identification of Protein-binding Chemicals. OECD Environment, Health and Safety Publications Series on Testing and Assessment No. 139. ENV/JM/MONO(2011).
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  12. Kato H, Okamoto M, Yamashita K, Nakamura Y, Fukumori Y, Nakai K, Kaneko H. 2003. Peptide-binding assessment using mass spectrometry as a new screening method for skin sensitization. J. Toxicol. Sci. 28: 19-24.
  13. Schultz TW, Yarbrough JW, Woldemeskel M. 2005. Toxicity to Tetrahymena and abiotic thiol reactivity of aromatic isothiocyanates. Cell. Biol. Toxicol. 21: 181-189.
  14. 14.0 14.1 Böhme A, Thaens D, Paschke A, Schüürmann G. 2009. Kinetic glutathione chemoassay to quantify thiol reactivity of organic electrophiles – Application to α, β-unsaturated ketones, acrylates, and propiolates, Chem. Res. Toxicol. 22: 742-750.
  15. OECD. Test No 442C: In chemico skin sensitisation: Direct Peptide Reactivity Assay (DPRA). 2015. OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects, OECD Publishing. Doi 10.1787/9789264229709-en.
  16. EURL ECVAM DB-ALM. Protocol No154: Direct Peptide Reactivity Assay for skin sensitisation testing. Available on: http://ecvam-dbalm.jrc.ec.europa.eu/.
  17. Yarbrough JW and Schultz TW. 2007. Abiotic sulfhydryl reactivity: A predictor of aquatic toxicity for carbonyl-containing α,β-unsaturated compounds. Chem. Res. Toxicol. 20: 558-562.
  18. Roberts DW and Natsch A. 2009. High throughput kinetic profiling approach for covalent binding to peptides: Application to skin sensitisation potency of Michael acceptor electrophiles. Chem. Res. Toxicol. 22: 592-603.
  19. Karlberg AT, Bergström MA, Börje A, Luthman K, Nilsson JL. 2008. Allergic contact dermatitisformation, structural requirements, and reactivity of skin sensitizers. Chem. Res. Toxicol. 21: 53-69.
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BASKETTER, D., POOLE, A. & KIMBER, I. 2017. Behaviour of chemical respiratory allergens in novel predictive methods for skin sensitisation. Regul Toxicol Pharmacol, 86, 101-106.

DIK, S., RORIJE, E., SCHWILLENS, P., VAN LOVEREN, H. & EZENDAM, J. 2016. Can the Direct Peptide Reactivity Assay Be Used for the Identification of Respiratory Sensitization Potential of Chemicals? Toxicol Sci, 153, 361-71.

HETTICK, J. M. & SIEGEL, P. D. 2011. Determination of the toluene diisocyanate binding sites on human serum albumin by tandem mass spectrometry. Anal Biochem, 414, 232-8.

HETTICK, J. M., SIEGEL, P. D., GREEN, B. J., LIU, J. & WISNEWSKI, A. V. 2012. Vapor conjugation of toluene diisocyanate to specific lysines of human albumin. Anal Biochem, 421, 706-11.

HOLSAPPLE, M. P., JONES, D., KAWABATA, T. T., KIMBER, I., SARLO, K., SELGRADE, M. K., SHAH, J. & WOOLHISER, M. R. 2006. Assessing the potential to induce respiratory hypersensitivity. Toxicol Sci, 91, 4-13.

HOPKINS, J. E., NAISBITT, D. J., KITTERINGHAM, N. R., DEARMAN, R. J., KIMBER, I. & PARK, B. K. 2005. Selective haptenation of cellular or extracellular protein by chemical allergens: association with cytokine polarization. Chem Res Toxicol, 18, 375-81.

LALKO, J. F., KIMBER, I., GERBERICK, G. F., FOERTSCH, L. M., API, A. M. & DEARMAN, R. J. 2012. The direct peptide reactivity assay: selectivity of chemical respiratory allergens. Toxicol Sci, 129, 421-31.

NATSCH, A. & GFELLER, H. 2008. LC-MS-based characterization of the peptide reactivity of chemicals to improve the in vitro prediction of the skin sensitization potential. Toxicol Sci, 106, 464-78.

WISNEWSKI, A. V., LIU, J. & REDLICH, C. A. 2013a. Connecting glutathione with immune responses to occupational methylene diphenyl diisocyanate exposure. Chem Biol Interact, 205, 38-45.

WISNEWSKI, A. V., MHIKE, M., HETTICK, J. M., LIU, J. & SIEGEL, P. D. 2013b. Hexamethylene diisocyanate (HDI) vapor reactivity with glutathione and subsequent transfer to human albumin. Toxicol In Vitro, 27, 662-71.